Construction of new cucumber fruit mottle mosaic virus derived subgenomic promotor and expression vector, and use thereof

ABSTRACT

The purpose of the present invention is to develop a cucumber fruit mottle mosaic virus vector so as to enable a study of gene function through gene silencing phenomenon in Cucurbitaceae plants, which has been difficult to study in the past, and to enable the study in fruits as well through stable expression. Furthermore, the present invention establishes a system that enables expression of a heterologous protein in plants, thereby, for the first time, providing a vector applicable to both the gene silencing phenomenon and protein expression in Cucurbtaceae plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase application filed under 35U.S.C. § 371 claiming benefit to International Patent Application No.PCT/KR2015/002358, filed on Mar. 11, 2015, which is entitled to priorityunder to Korea application no. 10-2014-0041785, filed Apr. 8, 2014.

The present invention was undertaken with the support of SupportingSystem Development of Molecular Breeding for Watermelon Seeds Exportusing Pathogen Resistance and Chromosomal Variants No. 213002041SBR10grant funded by the Ministry of Agriculture, Food and Rural Affairs(MAFRA).

TECHNICAL FIELD

The present invention relates to a new Cucumber fruit mottle mosaicvirus (CFMMV)-derived subgenomic promoter (SGP) and a CFMMV vectorincluding the promoter.

The present invention also relates to a composition which includes thevector for plant transformation, a host plant transfected by the vector,and a method of inducing gene silencing or gene expression in plantsusing the vector.

BACKGROUND ART

Today, an analysis of the nucleotide sequence of a plant is rapid due toadvancing sequencing methods and cost reduction. Among Cucurbitaceaeplants, recently, full-length sequences of cucumber (2009), melon(2012), and watermelon (2013) were disclosed, but, in construction of atransformant, there is a difficulty in conducting a study on genefunction through reverse or forward genetics due to a low success rateand inadequate development of a tool for a gene function analysis. Tosolve such a problem, viral vectors that can be studied are beingdeveloped. While viral vectors capable of being applied to other cropssuch as tomatoes, peppers, beans, etc. have been developed over time,the only choice for Cucurbitaceae plants is Apple latent stunt virus,which was developed in Japan in 2009. A vector constructed to express aheterologous protein in plants based on Tobamovirus, which infectscucurbits, is the Cucumber green mottle mosaic virus (CGMMV), which hadan expression result without a prediction of a subgenomic RNA promoter(SGP) with a small-sized epitope attached behind the coat protein.Therefore, to achieve gene silencing and stable heterologous proteinexpression, an attempt was made to construct a vector by isolating andidentifying CFMMV using Cucurbitaceae plants as main hosts. CFMMV is amember of the genus Tobamovirus, for which crops such as Cucurbitaceaeplants including cucumbers, pumpkins, melons, oriental melons,watermelons, gourds, and tobacco plants are hosts.

The inventors submitted the full-length sequence of CFMMV that had beenpreviously isolated and identified to the NCBI Genbank (Accession no.JN226146) and also received a patent for a recombinant clone of aCFMMV-derived attenuative virus (10-1342084). The present invention isfor expressing the whole or a part of an insert in a host plant byinserting an innate gene of the host plant through artificialmodification of the nucleotide sequence of CFMMV or inserting a gene ofa foreign organism.

Tobamovirus is characterized by having a single genome, whose subgroupsinclude Solanaceae-, Cruciferae- and Cucurbitaceae-infectiousTobamoviruses, and recently, Malvaceare-infectious andCucurbitaceae-infectious Tobamoviruses have been reported. Developmentand research on viral vectors using tobacco mosaic virus (TMV), which isa representative Tobamovirus, and tomato mosaic virus (ToMV), tobaccomottle green mosaic virus (TMGMV) and sunn-hemp mosaic virus (SHMV) havebeen successfully performed in tobacco and tomato.

In the development of viral vectors, it is important to select aninsertion position of a multiple cloning site (MCS), and, following theposition selection, subgenomic promoter (SGP) mapping should bepreferentially performed.

The inventors intend to provide a host plant-infectious viral vectorthat can be used in studying a gene function through gene silencing inCucurbitaceae plants, which is difficult, and can also in vivo express aheterologous protein in plants.

DISCLOSURE Technical Problem

An aspect of the present invention is to provide a new CFMMV-derivedsubgenomic promoter (SGP).

Another aspect of the present invention is to provide a CFMMV vectorincluding the promoter.

Still another aspect of the present invention is to provide a host planttransfected by a vector, and a transformed cell line.

Yet another aspect of the present invention is to provide a compositionincluding the vector or cell line for transformation and a method ofinducing gene silencing or gene expression in plants using the same.

Technical Solution

An aspect of the present invention provides a subgenomic promoter (SGP)consisting of a nucleotide sequence from −204 bp to +160 bp from thestart codon of the coat protein of CFMMV.

In the present invention, the CFMMV is a member of the Tobamovirus,which is, like other Tobamoviruses, a positive sense-single stranded(ss) RNA virus whose genetic information exists on RNA. The CFMMV genomeconsists of a nucleotide sequence of SEQ. ID. NO: 1.

The term “promoter” used herein refers to a DNA sequence capable ofregulating expression of a coding sequence or functional RNA. In thepresent invention, the promoter consists of a nucleotide sequence from−204 bp to +160 bp from the start codon of the coat protein, and thestart codon of the coat protein of the CFMMV may be located atnucleotides 5855 to 5857 of the CFMMV genome of SEQ. ID. NO: 1.

Also, the subgenomic promoter may consist of a nucleotide sequence fromone selected from the group consisting of −204 bp, −187 bp, −180 bp,−170 bp, −163 bp, −157 bp, −152 bp, −148 bp, −143 bp, −135 bp, −127 bp,−121 bp, −110 bp, −100 bp, −93 bp, −81 bp, −77 bp, −55 bp and −30 bp to+160 bp from the start codon of the coat protein and, most preferably,consists of a nucleotide sequence from −93 bp to +100 bp from the startcodon of the coat protein. The subgenomic promoter consisting of thenucleotide sequence from −93 bp to +100 bp from the start codon of thecoat protein may be represented by a nucleotide sequence of SEQ. ID. NO:2.

In one embodiment of the present invention, CFMMV is derived from a drymelon leaf provided from a Virus GenBank. The CFMMV was subjected tofull-length cloning to ensure an infectious full-length clone, and basedon the clone, construction of gene-insertable MCS was attempted.

In one embodiment of the present invention, the secondary structure ofsubgenomic RNA of CFMMV was predicted in order to find the range of SGPfor coat protein expression, and the SGP range showing the highestefficiency was provided through promoter mapping.

Also, one aspect of the present invention provides a CFMMV vectorincluding the subgenomic promoter.

The term “vector” used herein indicates serving to induce gene silencingin host plants or to deliver a nucleotide fragment for inducing geneexpression to a host plant, and the vector of the present inventionincludes a CFMMV-derived subgenomic promoter.

In the vector of the present invention, a nucleotide sequence forinducing gene silencing or gene expression is operably linked to theCFMMV-derived subgenomic promoter. The term “operably linked” usedherein refers to a functional linkage between a nucleic acid expressionregulatory sequence (i.e., promoter sequence) and a different nucleicacid sequence, and thus the regulatory sequence regulates transcriptionand/or translation of the different nucleic acid sequence.

In addition, the vector of the present invention includes at least onerestriction enzyme recognizing nucleotide sequence capable of cloning anucleotide sequence encoding a protein for expression, that is, amultiple cloning site (MCS) sequence. Restriction enzymes include, forexample, Eag I, EcoR I, EcoR II, BamH I, Bgl II, BstB I, Hind III, TaqI, Not I, Hinf I, Sau3A, Pac I, Pov II, Sma I, Hae III, Hga I, Alu I,EcoR V, EcoP15 I, Kpn I, Pst I, Sac I, Sal I, Sca I, Spe I, Sph I, StuI, Xba I, and Xho I, but the present invention is not limited thereto.

Also, the vector of the present invention may be additionally fused witha different sequence to facilitate purification of a recombinant targetprotein expressed from the vector, and the additional sequence may be,for example, a sequence encoding glutathione S-transferase (GST),maltose-binding protein (MBP), FLAG, 6× hexahistidine (His), Nutilization substance A (NusA) or thioredoxin (Trx). Due to theadditional sequence for purification, the protein expressed in a hostmay be rapidly and easily purified by affinity chromatography.

Also, the nucleotide sequence for inducing gene silencing in the vectorof the present invention may be RNA interference (RNAi), smallinterference RNA (siRNA), short hairpin RNA (shRNA), and micro RNA(miRNA), etc.

Also, the vector of the present invention may further include a CFMMV T7(SEQ. ID. NO: 5) or SP6 (SEQ. ID. NO: 6) promoter, or a CaMV 35Spromoter.

The CFMMV vector according to the present invention may effectivelyinduce gene silencing or gene expression in host plants. It can beapplied to study gene silencing and in vivo expression of a heterologousprotein and particularly may be more effectively utilized inCucurbitaceae plants.

Another aspect of the present invention provides a cell line transformedby the vector.

The cell line is not limited in type, and encompasses all cell lineswhich can infect plants. In an embodiment of the present invention,Agrobacterium was used.

Still another aspect of the present invention provides a host planttransfected by the vector or cell line.

The host plant may be a Cucurbitaceae plant. Examples of thenon-limiting Cucurbitaceae plants may include pumpkin, cucumber,watermelon, melon, oriental melon, gourd, and sponge gourd.

Yet another aspect of the present invention provides a compositionincluding the CFMMV vector or the cell line for plant transformation.

The composition may further include a P19 suppressor. The P19 suppressormay be RNA interference (RNAi), a small interference RNA (siRNA), shorthairpin RNA (shRNA), or micro RNA (miRNA), but the present invention isnot limited thereto.

The term “transformation” used herein refers to conversion of thephenotype of a host by injecting foreign DNA into host cells, andthroughout the specification, the transformation is substituted withvarious terms such as transfection, transformation, and transduction.

Yet another aspect of the present invention provides a method ofinducing gene silencing or gene expression in plants, the methodincluding inoculating a plant with the CFMMV vector or the cell line.

The method may include further inoculating the plant with a P19suppressor.

In an embodiment of the present invention, a CFMMV vector (p35sCF.001)including a CaMV 35S promoter, which facilitates transcription inplants, is provided. A vector for proliferating CFMMV was developed byinserting the same vector into an expression vector for transformationusing a transcription mechanism in plants.

In an embodiment of the present invention, a method of infectingp35sCF.001 with high efficiency is provided. When GV3101-pPM90 in theAgrobacterium family is transformed with p35sCF.001 and then theresulting transformant is injected into plant leaves, inoculationefficiency reaches approximately 100%. Also, the action of viralproliferation suppressing mechanism (defense mechanism) of plants inexpression of a heterologous protein significantly reduces an expressionlevel. Therefore, the present invention includes improving expression ofa heterologous protein and virus proliferation even in CFMMV byco-inoculation of p35sCF.001 with the P19 protein which was previouslyreported.

In addition, in one embodiment of the present invention, a method ofinserting an insert having the highest efficiency when applied in genesilencing is provided. Because of the characteristics of the vectorprovided from the present invention, for gene silencing, onlyapproximately 200 to 300 bp of a fragment is inserted, rather than awhole target gene. In this case, efficiency varies according to a geneposition and a method. When two adjacent fragments with the 5′-terminusand the 3′-terminus attached at both ends were inserted into a viralvector, the highest gene silencing efficiency was shown. In watermelon,a viral vector was stably retained until it grows up to get fruit, andgene silencing was also shown.

Advantageous Effects

The CFMMV vector according to the present invention may effectivelyinduce gene silencing or gene expression in host plants. It can beapplied to study the gene silencing and in vivo expression of aheterologous protein and particularly may be more effectively used inCucurbitaceae plants.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a prediction of the secondary structure of RNA for SGPmapping of a region downstream from the start codon of a coat proteinand a schematic diagram of a variant vector.

FIG. 2 shows EGFP gene expression according to the mapping of a regiondownstream from the start codon of a coat protein. A vector having +100bp of SGP showed high EGFP expression under blue light, which was alsoshown by Western blotting. Lane M-protein marker; Lane P—positivecontrol (CFMMV-Cm, N. benthamiana); Lane N—negative control (Mock); Lane1—pCF(+)68 bp-egfp; Lane 2—pCF(+)88 bp-egfp; Lane 3—2pCF(+)100 bp-egfp;Lane 4—pCF(+)127 bp-egfp; Lane 5—pCF(+)140 bp-egfp; Lane 6—pCF(+)160bp-egfp.

FIG. 3 shows a prediction of the secondary structure of RNA for SGPmapping for a region upstream from the start codon of a coat protein anda schematic diagram of a variant vector.

FIG. 4 shows the expression of a coat protein confirmed bySDS-polyacrylamide gel electrophoresis by inoculating N. benthamianawith clones constructed by SGP mapping of a region upstream from thestart codon of the coat protein and extracting a protein from theinoculated leaf thereof. It was confirmed that coat protein expressionoccurred in all samples except ΔMP-30 bp. Lane P—positive control(CFMMV-Cm, N. benthamiana); Lane N—negative control (Mock); Lane 30˜204bp (ΔMP-30 bp˜204 bp variant clone).

FIG. 5 is a schematic diagram completed by being constructed based onSGP mapping of a coat protein.

FIG. 6 shows EGFP expression confirmed by inoculating N. benthamianawith a vector having SGP from −93 bp to +100 bp and relativequantification of EGFP expression levels through real-time qPCR byextracting RNA of upper two leaves of N. benthamiana infected by thevector having −81, −100 or −110 bp of SGP with respect to −93 bp. It wasconfirmed that a pCF93-egfp-inoculated subject exhibited the highestmRNA expression level of EGFP.

FIG. 7 shows comparative expression levels of green fluorescent proteinsaccording to co-inoculation with P19. RNAs of whole inoculated leaves,inoculated upper second, third, and fourth leaves of N. benthamiana wereextracted to confirm the difference in mRNA expression levels of EGFPthrough real-time qPCR, and proteins were extracted to confirm thedifference in EGFP protein expression through western blotting.

FIG. 8 is a schematic diagram illustrating a position of inserted pdsgene and a cloning method.

FIG. 9 shows a relative quantification result to show the suppression ofpds gene mRNA expression in leaves by real-time qPCR by extractingphenotypes and RNAs of N. benthamiana, melon, and watermelon accordingto the suppression of pds gene expression. In watermelon, efficiencydifference is shown according to a gene amplification position and aninserting method.

FIG. 10 shows a relative quantification result obtained by semi qRT-PCRto show suppression of mRNA expression of pds gene in leaves byextracting phenotypes and RNAs from fruit flesh of three types ofwatermelon cultivars according to the suppression of pds geneexpression, and contents of β-carotene and lycopene, measured by HPLC.

FIG. 11 is a list of primers used in Real-time qPCR.

FIG. 12 is a schematic diagram of a CFMMV vector including T7 and SP6promoters.

FIG. 13 is an electrophoresis image of a product obtained by cloning ofa CFMMV vector including a T7 promoter and in vitro transcription, and aresult of egfp expression in inoculated leaves of N. benthamiana wheninoculated with the product.

MODES OF THE INVENTION

Hereinafter, the present invention will be described in further detail.Examples are merely provided to more fully explain the presentinvention, and it is obvious to those of ordinary skill in the art that,according to the gist of the present invention, the scope of the presentinvention is not limited to these examples.

<Example 1> Construction of CFMMV Vector

For gene insertion, MCS constructs which do not exist in virusesproliferated in the nature are required. Also, promoters capable ofexpressing a gene inserted into MCS are needed, and SGP is used,considering the features of the viruses. Since the SGP range ofCucurbitaceae-infectious Tobamovirus including CFMMV has not beenstudied, it will be provided in the present invention.

<Example 1-1> Prediction of Two-Dimensional Structure of SGP of CFMMVCoat Protein

A promoter-specific structure was identified using an RNA predictionprogram to utilize SGP of the coat protein, and then several estimatedranges were selected. An anti-sense RNA sequence from MP to CP wasinserted into an Mfold web server program, and an RNA secondarystructure at the lowest energy level was constructed (FIG. 1a ).

<Example 1-2> Promoter Mapping from CFMMV Clones (Mapping of DownstreamRegion of Start Codon)

SGP exists upstream and downstream with respect to the start codon (ATG)of the coat protein. To find the range of an operated promoter, the coatprotein was substituted with enhanced green fluorescence protein (EGFP)which is able to facilitate visual detection of protein expression, andeach of vectors including the range of +68, +88, +100, +127, +140 and+160 bp from the start codon of the coat protein was cloned (FIG. 1b ).Here, the start codon of the coat protein in changed from ATG to ACG.The resulting viral vector was transfected into Agrobacterium, which wasinjected into N. benthamiana to observe EGFP expression in theinoculated leaves. It was confirmed under blue light that subjectsinoculated with CFMMV vectors with +100 bp at 6 dpi showed the highestprotein expression. Six leaf discs were equally harvested from theagroinfected ranges of inoculated leaves to extract proteins. Equivalentamounts of the proteins were analyzed by electrophoresis and westernblotting, and the results thereof are shown in FIG. 2.

As shown in FIG. 2, EGFP protein expression was detected only from +100bp inoculated subject.

<Example 1-3> Upstream Mapping of Start Codon of CFMMV Coat Protein

To determine the downstream promoter range from the start codon, a partof the downstream of the start codon of the coat protein was removed(movement protein-encoded region), and the expression of the coatprotein was identified. 13 mutants from −204 to −30 bp upstream thestart codon of the coat protein were constructed (ΔMP-30 bp˜204 bp, FIG.3), and each was injected into leaves of N. benthamiana. Six leaf discswere equally harvested from the agroinfected regions of the inoculatedleaves so as to extract proteins, which were identified throughSDS-PAGE. The results are shown in FIG. 4.

As shown in FIG. 4, expression of all proteins except ΔMP-30 bp wasidentified.

<Example 2> Construction of CFMMV Vector Through the Most Efficient SGPSelection

Based on the mapping results, while downstream +100 bp was fixed,vectors having 19 different (−30 bp, −55 bp, −77 bp, −81 bp, −93 bp,−100 bp, −110 bp, −121 bp, −127 bp, −135 bp, −143 bp, −148 bp, −152 bp,−157 bp, −163 bp, −170 bp, −180 bp, −187 bp, −204 bp) SGPs wereconstructed and injected into N. benthamiana, followed by selection of avector having SGP showing the highest efficiency. As a result, thehighest EGFP expression was shown from the vector having SGP from −93 bpto +100 bp (pCF93-egfp).

<Example 3> Sequencing for Selected CFMMV Vector

A nucleotide sequence of the selected vector pCF93-egfp was analyzed bySolGent, and the result is set forth in SEQ. ID. NO: 3.

<Example 4>Agrobacterium Transformant for CFMMV Vector Inoculation andInoculation

Among inoculation methods with a CFMMV vector having a 35S promoter, themost effective method is agroinfection. 50 ng of the vector wastransformed to Agrobacterium-family GV3101 (or GV3101-pPM90) by anelectric shock, and the resultant cells were grown in a 5-ml tubecontaining LB medium by shaking the culture at 28° C. for approximately16 hours and then subcultured in 30 ml LB medium. Here, 0.01 M MES (pH5.6) and 20 μM acetosyringone were added to 30 ml LB medium and culturedwith shaking at 28° C. up to O.D₆₀₀=0.8˜1.0, followed by collecting thecells by centrifugation at 6000 rpm for 5 minutes. Inoculants (4.4 g/Lof MS salt, 0.01M MES (pH 5.6), 2% sucrose, 200 μM acetosyringone) wereadded and diluted to activate viruses at room temperature over 4 hours.The activated viruses were injected into the backside of leaves using 1ml syringe without a needle.

<Example 5> RNA and Protein Extraction

Following grinding the harvested sample in liquid nitrogen, 800 μlreagent (MRC) prepared by adding 8 μl mercaptoethanol to the powder wasadded and stirred at 65° C. for 5 minutes. The resulting product wascentrifuged at 13000 rpm for 3 minutes to obtain a supernatant, whichwas transferred to a new tube, treated with 300 μl chloroform, andvortexed for 15 seconds. The resultant product was centrifuged at 13000rpm and 4° C. for 15 seconds, and then a supernatant was transferred toa new tube. An equivalent amount of isopropanol was added to the tubeand precipitated at −20° C. for 20 minutes. A washing procedure, whichincludes centrifugation of the pellets at 13000 rpm and 4° C. for 10minutes, discarding of isopropanol, addition of 1 ml of 70% ethanol, andthen centrifugation of the resulting solution at 13000 rpm and 4° C. for10 minutes, was performed twice. After remaining ethanol was completelyremoved, the pellets were dissolved in 30 μl water and treated withDNase I, thereby isolating only pure RNA.

Leaf samples were ground in liquid nitrogen. 900 μl of RNA extractionbuffer was added to a 2 ml tube and stirred well, followed by additionof 900 μl phenol and gentle vortexing. The resulting product wascentrifuged at 13000 rpm and 4° C. for 15 minutes to recover anintermediate protein layer. The above procedure was purified through PCItreatment twice to recover a protein. The protein was precipitated in100% acetone at −20° C. for approximately 1 hour and isolated bycentrifugation at 10000 g and 4° C. for 10 minutes, followed bycompletely discarding acetone and then washing with 80% acetone severaltimes. The recovered protein was naturally dried for approximately 30minutes and then dissolved in 100 μl of 1% SDS.

<Example 6> Real-Time qPCR for Confirming CFMMV Vector Efficiency

From N. benthamiana infected by a vector having −81 bp, −100 bp or −110bp of SGP with respect to −93 bp, which showed the highest expression ofa green fluorescent protein as visually detected, RNAs of inoculatedupper second leaves were extracted, and in order to synthesize cDNA fromisolated and purified RNA, RT-PCR was performed with 20 μl of a reactionmixture including 2 μg of total RNA, 1× buffer, 10 mM dNTP, 0.05M DTT,40 U RNase Inhibitor, and 200 U Superscript III reverse transcriptaseusing 100 ng of a random hexamer. For comparative quantification usingcDNA 1/10 diluent as a template, a primer for GAPDH gene was constructedto be used in normalization between samples. To quantify a target gene,EGFP-specific primers were used, and the primers are shown in FIG. 11.

Real-time qPCR was performed using a final 20 μl reaction mixtureincluding the following substances: 2 μl of the first chain cDNAs, 2×master mix, and 20× Evagreen dye (Biofact Co.). PCR was performed underthe following temperature conditions: 40 cycles of 94° C. for 12minutes, 94° C. for 10 seconds, and 60° C. for 30 seconds. According tothe PCR results, it was confirmed that the 35SCF-93 bp::EGFP inoculatedsubject maintains the highest expression level of EGFP mRNA (FIG. 6).

<Example 7> Comparison of Expression Levels of Heterologous Protein inCo-Inoculation with P19 Suppressor

In single-inoculation with pCF93-egfp vector and co-inoculation with aP19 suppressor and a vector, EGFP expression levels in inoculated leavesand upper leaves were determined on RNA and protein levels. The P19suppressor serves to suppress post-transcriptional gene silencing (PTGS)and particularly serves to maintain virus proliferation without damageby preventing degradation of a dsRNA-type viral product formed in avirus proliferation process by a plant preventive mechanism. Therefore,in this study, the P19 suppressor was used to maintain the highexpression level of EGFP mRNA included in the virus, as well as thevirus, and thus increase the expression level of a heterologous proteinin plants. Co-inoculation was performed by transforming a vectorincluding a 35S promoter and a P19 gene into Agrobacterium GV3101 andinjecting a mixture of the resulting inoculant and pCF93-egfp at a ratioof 1:1. From inoculated leaves (1st) and inoculated upper second (2nd),third (3rd) and fourth (4th) leaves, six leaf discs were harvested toextract both RNA and proteins according to the method described inExample 5, and protein expression levels were visualized by westernblotting (disclosed in Example 8). Also, as disclosed in Example 6, cDNAwas synthesized and analyzed under the same condition and compositionthrough real-time quantitative PCR to compare mRNA expression levels ofegfp. In the single-inoculation with a vector, when a fluorescence valueof the egfp expression level of the inoculated leaves was set as 1 todigitize a relative quantification value, the sum of expression levelsfrom four leaves was 374.97, and the sum of expression levels from fourleaves when co-inoculated with a P19 suppressor was 787.09. In theco-inoculation with the P19 suppressor, an approximately two-foldincrease in egfp mRNA was shown (FIG. 7).

<Example 8> SDS-PAGE and Western Blotting for Confirming GreenFluorescent Gene Expression

A protein sample was isolated from an SDS-polyacrylamide gel, and anitrocellulose (NC) membrane was transferred by electroblotting using anelectrotransfer apparatus (Bio-Rad, USA). A blotted membrane wascarefully separated and transferred to an SNAP-id system (Millipore Co.)to bind antibodies to proteins on the membrane according to the sequenceof blocking, 1^(st) antibody (1:1000 GFP, Clontech Co.) binding,washing, 2^(nd) antibody (1:7500; Anti-Rabbit IgG Ap conjugate, PromegaCo.) binding, and washing. The membrane on which the binding wascompleted was reacted with an alkaline phosphatase (AP) solution (100 mMTris-Cl, pH8.0; 100 mM NaCl, 5 mM MgCl₂) for 1 minute, and 1 mL ofWestern Blue stabilized substrates (Promega Co.) with respect to thealkaline phosphatase was added.

<Example 9> Gene Silencing Using CFMMV Vector

Gene silencing was observed in Cucurbitaceae plants by inserting a partof a target gene into vectors having SGP of the coat protein, which weredifferent from the selected vector.

<Example 9-1> Cloning and Insertion of Target Gene

As a target gene, a Phytoene desaturase (pds) gene, which forms aβ-carotene synthesis pathway, was selected. cDNA was synthesized from 2μg of RNA extracted from each plant using superscript III reversetranscriptase and oligo dT primers and cleaved with XhoI-PmeI to form200 to 300 bp of an insert, and then the insert was inserted into avector. Also, according to previous reports showing that gene silencingcaused by viruses was caused by the position and size of the insertedgene, the pds gene of melon disclosed in the NCBI Genbank was 1904 bp, aprimer capable of constructing a fragment by attaching a region near the5′-terminus, a region near the 3′-terminus, and genes in two regions ina coding region even in watermelon was constructed with reference to thenucleotide sequence of the pds gene to be used to amplify cDNAsynthesized from RNA of each plant, the cDNA was also cleaved withXhoI-PmeI, and each resulting fragment was inserted and inoculated,thereby confirming efficiency of gene silencing (FIG. 8).

<Example 9-2> Application of Gene Silencing to Leaves

Due to the decreased expression level of the pds gene, phenotypes ofwhitening of leaves by photo-bleaching were identified in N.benthamiana, melon, cucumber and watermelon were confirmed, anddifferent results were shown according to a gene insertion method.Whitening caused by pds gene silencing was maintained until the growthwas completed (FIG. 9).

<Example 9-3> Application of Gene Silencing to Fruit

A Pds gene encodes an enzyme constituting a β-carotene biosynthesispathway, which is an early-stage enzyme of lycopene. Therefore, due tosilencing of the pds gene, whitening of red fruit flesh, withoutlycopene, in watermelon was observed, and positive results were obtainedfrom all of the three types of cultivars. Accordingly, a result whichcan be stably applied is provided as a means for gene function analysisin fruits (FIG. 10).

<Example 9-4> Real-Time qPCR for Confirming Gene Silencing at RNA Level

A whitened (photo-bleached) leaf sample was harvested to extract RNA,and cDNA was synthesized using superscript III reverse transcriptase andoligo dT, and the suppression of pds gene expression was confirmedthrough real-time qPCR. As a gene for normalization, 18S rRNA gene wasused in Cucurbitaceae plants, and a primer was constructed from a partof the pds gene to be used in a comparative quantification. Thereal-time qPCR was performed using total 20 μl of a reaction mixtureincluding the following substances: 2 μl of first chain cDNAs, 2× mastermix, and 20× Evagreen dye (Biofact Co.). PCR was performed under thefollowing temperature conditions: 40 cycles of 94° C. for 12 minutes,94° C. for 10 seconds, and 60° C. for 30 seconds. As a result, it wasconfirmed that gene expression levels were decreased in all plants, andwhen an experiment according to a method of inserting a target genefragment in watermelon was performed, gene expression was mostefficiently suppressed in a subject inoculated with fragments at bothpositions (FIG. 9).

<Example 9-5> Analyses of Lycopene and Beta-Carotene Contents in Fruits

45 dap (day after pollination) of watermelon was harvested to obtain 15g of fruit flesh, followed by instantly freezing in liquid nitrogen andfreeze-drying for one week.

1. Pretreatment of Samples

0.1 g of watermelon cut finely using a blender was put into a screw taptube, and then beads and samples were added at a ratio of 1:1. Followingaddition of 1 ml of 0.5 mM BHT-added ethanol, the resulting mixture wasput into a bead buffer and then vigorously stirred for 2.5 minutes. Theethanol, sample, and beads in the tube were all transferred to a 15 mltube, and everything left in the screw tap tube was washed with 1 mlacetone three times and transferred to a 15 ml tube. 3 ml of petrolether was added to the tube and vortexed, followed by adding 8 ml of 20%NaCl and vortexing. The resulting mixture was centrifuged at 3000 rpmfor 10 minutes, and only the supernatant was harvested. After theincrease in a sample mass, Na₂SO₄ was added to the sample, and then thesample was passed through a filter (PTFE, 13 mm, 0.2 μm; Advantec,U.S.A.) to finally prepare an analyte.

2. Quantitative Analysis of Carotenoid Using Liquid Chromatography

A carotenoid content was quantified using a liquid chromatography system(Waters 2489; Waters, U.S.A.) equipped with a reversed-phase column(Kinetex 2.6 μm, C18 100 A, 100×4.60 mm; Phenomenex, U.S.A.). Mobilephase A was 78% methanol, and mobile phase B was 100% ethyl acetate.Separation conditions included 0-10 min, 70% B; 10-14 min, 100% B;14-14.01 min, 0% B; and 14.01-20 min, 0% B, and a flow rate was 1 ml perminute. As a standard for quantification, β-carotene and lycopene(Sigma-Aldrich Co., U.S.A.) were used, and optical densities weremeasured at 450 nm and 660 nm to quantify the total content ofcarotenoid.

From three types of cultivars such as pCF93-pds-infected chris cross,DAH and 2401, it was confirmed that the accumulation of β-carotene andlycopene was decreased by a factor three to 110 compared to that ofCFMMV-infected watermelon (FIG. 10).

<Example 10> Construction and Inoculation of CFMMV Vector Including T7Promoter and SP6 Promoter

CP SGP of the virus was first designed in the range of +55 bp based on a2D structure result. Using pT7CF-Cm_(flc) as a basic backbone, CP wasremoved, 3′NTR was primarily inserted to create MluI and PmeI sites,and, while the initiation of translation was suppressed by substitutingthe start codon ATG into AAG to determine the SGP range, the createdgene was amplified by PCR and inserted to include only a desired rangeof SGP to prepare Pad. Accordingly, MCS including PacI-MluI-PmeI wasconstructed, a SphI-linearized vector was inoculated through in vitrotranscription by inserting EGFP with the Pad and PmeI previously formedusing a mMASSAGE mMACHINE kit (Ambion). In order to construct a vectorincluding the coat protein, a vector using an upstream subgenomicpromoter range of +100 bp and including a subgenomic promoter rangecorresponding to −55 bp to −204 bp from the start codon of the coatprotein was constructed, and in this process, like the structure of avector including a 35S promoter, restriction sites were changed toXhoI-PmeI, and the sequence of the start codon was also changed toATG→ACG to construct a vector. The result and structure of the vectorare shown in FIGS. 12 and 13.

The invention claimed is:
 1. A nucleic acid molecule comprising asubgenomic promoter comprising the nucleotide sequence from −204 bp to+160 bp from a start codon of the coat protein of Cucumber fruit mottlemosaic virus (CFMMV) operably linked to a heterologous nucleotidesequence.
 2. The nucleic acid molecule of claim 1, wherein the CFMMVgenome comprises the nucleotide sequence of SEQ. ID. NO:
 1. 3. Thenucleic acid molecule of claim 1, wherein the start codon of the coatprotein of the CFMMV is located at nucleotides from 5855 to 5857 of theCFMMV genome of SEQ. ID. NO:
 1. 4. The nucleic acid molecule of claim 1,wherein the subgenomic promoter comprises a nucleotide sequence from oneselected from the group consisting of −204, −187, −180, −170, −163,−157, −152, −148, −143, −135, −127, −121, −110, −100, −93, −81, −77, −55and −30 bp to +160 bp from the start codon of the coat protein.
 5. Thenucleic acid molecule of claim 1, wherein the subgenomic promotercomprises the nucleotide sequence from −93 bp to +100 bp from the startcodon of the coat protein.
 6. The nucleic acid molecule of claim 5,wherein the subgenomic promoter comprises the nucleotide sequence ofSEQ. ID. NO:
 2. 7. A Cucumber fruit mottle mosaic virus (CFMMV) vector,comprising the nucleic acid molecule of claim
 1. 8. The CFMMV vector ofclaim 7, wherein the vector shows a gene silencing or gene expressioneffect in Cucurbitaceae plants.
 9. The CFMMV vector of claim 7, whereinthe vector comprises the nucleotide sequence of SEQ. ID. NO:
 3. 10. TheCFMMV vector of claim 7, wherein the vector further comprises a T7, SP6or 35S promoter.
 11. A cell line which is transformed by the vector ofclaim
 7. 12. A host plant transfected by the vector of claim
 7. 13. Thehost plant of claim 12, wherein the host plant is Cucurbitaceae plant.14. A composition for transforming a plant, comprising the Cucumberfruit mottle mosaic virus (CFMMV) vector of claim
 7. 15. The compositionof claim 14, wherein the composition further comprises a P19 suppressor.16. A method of inducing gene silencing or gene expression in a plant,comprising inoculating the plant with the Cucumber fruit mottle mosaicvirus (CFMMV) vector of claim
 7. 17. The method of claim 16, wherein themethod comprises further inoculating the plant with a P19 suppressor.18. A host plant transfected by the cell of claim
 11. 19. A compositionfor transforming a plant, comprising the cell of claim
 11. 20. A methodof inducing gene silencing or gene expression in a plant, comprisinginoculating the plant with the cell of claim 11.